Effects Of Ep400 On The Expression Of Hpv18 E6-E7 And The Growth Of Cervical Cancer Cells

Cervical cancer is the second common malignant tumor that threatens womans health worldwide.The leading cause of cervical cancer is the persistent infection of cervical epithelial cells by the high-risk type of human papilloma viruses(HPV).HPV18 is a type of high-risk HPV and has been confirmed to be correlated with cervical cancer.In HeLa cells,HPV 18 DNA fragments integrate into the host genome and HPV 18 E6-E7 gene expression is under the regulation of cellular transcription factors.EP400 is a cellular transcription co-factor with ATPase activity.It was reported to inhibit the HPV 18 E6-E7 gene in an E2-dependent manner.In this study,we would investigate the influence of EP400 on HPV18 E6-E7 gene regulation in HeLa cells which are E2-deficient.Firstly,HeLa cells were transfected with the EP400 specific siRNA,the expression of HPV 18 E6-E7 was determined using RT-PCR,Real-time PCR and Western blot analysis.Then we overexpressed HPV 18 E6-E7 in HeLa cells,using RT-PCR,Real-time PCR and Western blot methods,the expression of E6-E7 was measured.The results showed that depletion of EP400 upregulated the transcription and expression of HPV 18 E6-E7 however overexpression of EP400 inhibited the expression of HPV 18 E6-E7 at both mRNA and protein levels.Secondly,in order to investigate the mechanism of EP400 inhibiting HPV 18 E6-E7,Luciferase reporter assays were performed with HPV18-LCR luciferase plasmids,and the results confirmed that EP400 has an inhibitory effect on HPV 18 promoter.To further explore the mechanism,chromatin immunoprecipitation experiments were carried out with RNA polymerase II antibodies or antibodies against histone H3 or H4.After the interference of EP400,the recruitment of RNA polymerase II on HPV 18 LCR increased.Meanwhile,the association histone H3 and H4 decreased.These results suggest that EP400 can affect nucleosome density,thereby affecting the RNA polymerase II recruitment on HPV 18 long control region.Taken together,the above results indicate that EP400 could inhibit HPV18 E6-E7 gene in an E2-independent way.Additionally,the effects of EP400 on HeLa cell proliferation and migration were investigated.At first,the EP400 was interfered,and cell proliferation and migration were detected by MTT,scratch and Transwell experiments.Then in order to further verify the experimental results,this study used overexpression of EP400 with the same method.The results showed that EP400 could inhibit the migration HeLa cells,and the effect on cell proliferation was not obvious.